One type of ligand-receptor assay is an immunoassay. Various known formats exist for immunoassays, including immunochromatographic test strips for detecting analytes in liquid samples. One format of a ligand-receptor assay uses a direct binding “sandwich” assay, wherein the analyte is bound by two specific binding molecules, the most common type of which is an antibody. Examples of this format are described in U.S. Pat. No. 4,861,711; H. Friesen et al. (1989), which discloses a solid-phase diagnostic device for the determination of biological substances; U.S. Pat. No. 4,740,468; L. Weng et al. (1988) which discloses a solid phase specific binding method and device for detecting an analyte; U.S. Pat. No. 4,168,146; A. Grubb et al. (1979) which discloses a solid phase method and strip with bound antibodies and U.S. Pat. No. 4,435,504; R. Zuk (1984) which discloses a chromatographic immunoassay employing a ligand-binding molecule and a label conjugate.
In one type of this format, described in U.S. Pat. No. 4,959,307; J. Olson (1990), the result is revealed as two lines (positive result) or one line (negative result).
One problem with the dominant format of sandwich-type chromatographic test strips is that antibodies are necessarily bound to a surface when encountering the analyte. Antibodies are covalently or non-covalently bound to the surface of colored particles as well as to fibers of a test strip membrane. These surfaces are not identical in composition or in shape. Attachment of an antibody to either surface is generally deleterious to that antibody's ability to bind antigen, for many reasons including 1. any method of attachment can cause steric hindrance, or blocking of access by the antigen to the antibody's binding sites, 2. deformation of the secondary, tertiary, and quarternary structure of the antibody molecule by the surface causes deleterious changes to the antibody's binding site, 3. dramatically reduced freedom of movement of the antibody in solution decreases the probability kinetics of successful antibody-antigen dockings, 4. non-covalent attachment through hydrophobic-hydrophilic interactions may result in a most-favored configuration that leaves the antibody binding site facing the surface as opposed to facing outward, thus completely hiding the binding sites, 5. covalent attachment adds substantial modifications to the structure of the antibody and may cause even more significant deformations than non-covalent binding, 6. while most antibodies are developed on the basis of optimal binding to antigens while freely dissolved in aqueous solutions, a different kind of binding is required when the antibody is surface-bound, and 7. binding of antibodies to surfaces may change the antibodies' affinity and/or specificity.
These problems are magnified in cases where the concentration of analyte in the sample is low, the affinity or on-rate of the antibody-antigen interaction is suboptimal, or when the samples are complex and present significant potential interferences for the antibody-antigen interaction. Especially in these cases, the decreased probability of a correct antibody-antigen docking can decrease the sensitivity of the assay, increase the background or cross-reactivity, or all of the above.
The second problem associated with the dominant format of the chromatographic test strip is that the format is not well-suited for assays where it is desired to detect any member of a set of analytes. An example of such an assay is a test for bacterial contamination, in which the presence of any of a large number of different bacteria would indicate contamination. In these assays, the optimal design would yield a positive result when any of the analytes were detected or when a group of analytes were detected, without the need to identify which specific analyte was present. An important subset of this kind of assay is a test that shows a positive result when the total number or concentration of several analytes exceeded a threshold level, without regard to the representation of any of the individual analytes within that total number.
In order to create such an assay using the dominant format of the chromatographic test strip, the manufacturer would have to choose several analyte-specific antibodies or pairs of antibodies, couple each to both the signal particles and the membrane, and then carefully calibrate the amounts and ratios of each set to ensure conformity with the sensitivity requirements of the assay. Relative sensitivities of the assay for each analyte are not changeable once the reagent ratios are set. It is exceedingly difficult to fine-tune the affinities, surface binding properties, and total amounts of each reagent in a predictable manner to achieve the performance requirements for a test strip that has a “total number” threshold of several analytes.
In addition, manufacture and quality control of each antibody-signal particle and each antibody on the membrane is considerably laborious and costly for all makers of sandwich-type chromatographic test strips. With multiple such components in a single test strip, this process would dramatically increase manufacturing costs and failure rates of strip tests. Since the failure of one component forces the manufacturer to fail the entire assembly, the cost of manufacture of a multi-analyte test strip is elevated in comparison to a single-analyte test strip.
Furthermore, it is difficult for a maker of a multi-analyte, multi-antibody-pair sandwich-type chromatographic test strip in the dominant format to change the number of analytes or the sensitivity of the test for any individual analyte. The new sensitivity profile necessitates a re-design and re-calibration of each component, especially if new antibody pairs are added.
Partially due to these difficulties, most of the marketed sandwich-type chromatographic test strips are single-analyte assays. In some cases, cross-reactivity of a single polyclonal or monoclonal antibody may enable a single test strip to detect more than one analyte.
Therefore, a need exists for assay that eliminates or reduces one or more of the current drawbacks.